The objectives of this project, and related progress in the past year, are reviewed below. (1) Development of a reverse genetics system that can be used to modify the antigenicity and virulence of rotaviruses (RVs) and to develop new vaccine candidates. Four years ago, Komoto et al (2006) described a helper virus-dependent, single-gene replacement reverse genetics system for RV. This system uses a replication-deficient vaccinia virus (rDIs-T7pol) as the source of T7 RNA polymerase (T7pol) to drive the transcription of the RV VP4 gene (SA11 strain) from a transfected cDNA plasmid. Infection of the transfected cells with a helper RV (KU strain) allowed the recovery of recombinant viruses that had incorporated the SA11 VP4 gene into the KU genome. While representing an important scientific breakthrough, application of the Komoto system is limited due to its inefficiency in generating recombinant viruses. To expand the usefulness of RV reverse genetics, we developed a novel method to recover single-gene recombinant viruses in which two independent mechanisms act on a single viral gene to select for the recombinant. We have coupled a mutant SA11 RV bearing a temperature-sensitive (ts) defect in the viral NSP2 protein with RNAi-mediated degradation of NSP2 mRNAs to isolate a virus bearing a single recombinant gene that evades both selection mechanisms. Recovery of recombinant viruses by this system is rapid and highly efficient. We have used our reverse genetics method to manipulate the RV genome by generating a panel of viruses with chimeric NSP2 genes. Combining characterized RV ts mutants and validated siRNA targets may permit the direct extension of this two-hit reverse genetics methodology to allow manipulation of other genes. (2) Identification of RV virulence determinants that antagonize host immune responses, notably the interferon signaling pathways. Earlier studies involving limited numbers of RV strains showed that the viral gene 5 product, NSP1, can antagonize interferon (IFN-a/b) expression by inducing the degradation of IFN-regulatory factors (IRF3, IRF5, IRF7) or a component of the E3 ubiquitin ligase complex responsible for activating NF-kB (b-TrCP). To gain a broader perspective of NSP1 activities, we examined various RV strains for the ability to inhibit IFN expression in cultured cells. We found that all strains encoding wild-type NSP1 impeded IFN expression, but that this activity was not strictly linked to IRF3 degradation. To identify other degradation targets involved in suppressing IFN expression, we used transient expression vectors to test the ability of a diverse collection of NSP1 proteins to target IRF3, IRF5, IRF7, and b-TrCP for degradation. The results indicated that human RVs rely predominantly on NSP1-induced degradation of IRF7 to suppress IFN signaling, whereas NSP1 proteins of animals RVs tend to target both IRF3 and IRF7, allowing the animal viruses a broader attack on the IFN signaling pathway. The results also indicated that NSP1-induced degradation of b-TrCP is an uncommon mechanism of subverting IFN signaling, but one that can be shared with NSP1 proteins inducing degradation of IRFs. Our analysis reveals that the activities of NSP1 proteins are diverse, with no obvious correlations between the degradation of any pair of target proteins. Thus, RVs have evolved distinct approaches for subverting the host antiviral response, a property consistent with the immense sequence variation noted among NSP1 proteins. (3) Analysis of the diversity and evolution of the RV genome. The scarcity of complete genome sequence information for the RVs prevents a comprehensive molecular analysis of RV diversity and evolution, limits our capacity to assess the impact of RV vaccines on the genetic make-up of viruses circulating in the human population, and undermines the full potential of reverse genetics systems. To address the need for additional sequence information, we have initiated several large scale genomic sequencing projects. (a) An important question is whether the widespread use of the RotaTeq and Rotarix vaccines will induce antigenic and/or genetic changes in commonly circulating RVs or will lead to the emergence of new G-type strains not covered by the vaccines. To address this question, we initiated a project to sequence the complete genomes of RVs collected from sick children at Vanderbilt Medical Center during the 2005-06 to 2008-09 winter seasons. The four seasons covered by these samples are particularly significant as they include seasons prior to and after introduction of the RotaTeq vaccine. Once completed, the sequencing data will be examined for evidence that vaccine usage induces shifts in the equilibrium of co-circulating G/P-type viruses and changes to the antigenic epitopes of the VP4 and VP7 proteins of circulating strains. (b) To gain insight into the diversity and evolution of human RVs circulating in a single location over a long period of time, we are in the process of analyzing the genomic sequences of G1, G2, G3, and G4 RVs isolated from sick children during 1974 to 1991 at Children's Hospital National Medical Center, Washington DC. Sequencing of the G2, G3, and G4 samples was completed. Results have revealed that genetically-distinct RV clades of the same G/P-type, but with varying neutralization epitopes, can co-circulate and cause disease. The findings also indicate that although genome reassortment can occur among RVs, most reassortant strains are replaced overtime by lineages with preferred gene constellations. (4) Analysis of the genetic make-up and stability of RV vaccine candidates. (a) Dr. Albert Kapikian and colleagues have developed a multivalent RV vaccine (UK BRV) from reassortment of antigenically-distinct human RVs with the bovine UK RV. The UK BRV vaccine includes six reassortants, differing in serotype (G1, G2, G3, G4, G8, G9). Master seed lots were prepared for each reassortant by the NIH (NIH Master Seeds) and distributed to companies interested in producing RV vaccines at low cost in developing counties. As part of their program to develop the vaccine, two companies have generated their own Master Seeds (Shantha, India, and WIBP, China) from the NIH Master Seeds, and then passed their Master Seeds subsequently to produce Working Batches and Production Batches of each viral component of the UK BRV vaccine. To support development of the vaccine, we have agreed to sequence the viral genomes in the Master Seeds and the Working and Production Batches produced by the companies. This information is necessary to provide safety information concerning the make-up and genetic stability of the viruses in the vaccine. In the past year, we completed sequencing efforts on the Shantha samples. In the coming year, we anticipate completing sequencing of WIBP materials. (b) RVs cause severe gastroenteritis in infants and young children;yet, several strains have been isolated from newborns showing no signs of clinical illness. Two of these neonatal strains, RV3 (G3P6) and 116E (G9P11), are being developed as live-attenuated vaccines. In this study, we sequenced the genomes of cell culture-adapted RV3 and 116E and compared their genes and protein products to those of other RVs. Using amino acid alignments and structural predictions, we identified residues of RV3 or 116E that may contribute to attenuation or influence vaccine efficacy. We also discovered residues of the VP4 attachment protein that correlate with the capacity of some P6 strains, including RV3, to infect newborns versus older infants. The results of this study enhance our understanding of the molecular determinants of RV3 and 116E attenuation and are expected to aid in the ongoing development of these vaccine candidates.